Summary Evidence of persistent viral genomes in humans after infection with acute RNA viruses is increasing as technological advances with enhanced sensitivity permit the detection of viral reservoirs. Such evidence challenges the dogma that acute viruses are completely cleared by the immune system. In parallel, evidence of a role for persistent viruses in promoting disease is growing. For example, persistent respiratory syncytial virus in humans is associated with asthma, persistent coxsackie virus causes cardiac disease, and persistent measles virus or West Nile virus cause encephalitis. Persistent viral genomes are also a major obstacle for eradicating virus from infected individual following antiviral therapies. Despite much speculation, the mechanisms driving the generation and maintenance of long-term viral reservoirs during infections with RNA viruses are completely unknown. Resolving these mechanisms is critical for limiting or eliminating persistent viral reservoirs and for reducing their impact in disease. Evidence suggests that defective viral genomes (DVGs) that are generated during viral replication are pivotal in the generation of persistent viral reservoirs. Specifically, we observed that while some cells become enriched in DVGs upon virus infection, others maintain a predominantly full-length viral genome content. Furthermore, despite showing initial potent antiviral responses, cells dominated by DVGs survive the infection and generate long-term viral reservoirs, while cells dominated by full-length viral genomes died. In this exploratory proposal we take advantage of powerful technology to differentiate full-length viral genomes from DVGs in infected cells to 1.Identify cellular mechanisms mediating the persistence of infected cells dominated by DVGs and 2. Identify and characterize in vivo viral genome reservoirs. Overall, we expect to establish the role of DVGs in the generation of long-term viral reservoirs, break ground in our understanding of the mechanisms promoting survival of cells destined to allow virus persistence, and determine if pathways driving persistently infected cells in vitro are also active in vivo.